Sensor, detection method and detection device

Abstract

A biosensor 1, which is an embodiment of a sensor according to the present invention, is provided with: a sensor element 10; and probe molecules 20 immobilized on the surface of the sensor element 10, and a marker substance 30 for estimating the amount of probe molecules 20 immobilized on the sensor element 10 is modified with the probe molecules 20.

Description

[Technical field]

[0001] The present invention relates to sensors such as biosensors, pH sensors, ion sensors, etc. Furthermore, the present invention relates to a detection method and a detection device using the above sensors. [Background technology]

[0002] There is a demand for highly sensitive detection methods that can rapidly detect substances to be detected (also called target molecules) such as cells, microorganisms, viruses, proteins, nucleic acids (DNA and RNA), peptides, amino acids, and ions.

[0003] In recent years, sensors using probe molecules that specifically interact with target molecules have been proposed. Known examples of such sensors include field effect transistor (FET) type, quartz crystal microbalance (QCM) type, and surface plasmon resonance (SPR) type sensors.

[0004] In the above-mentioned sensor, the probe molecule is immobilized on the surface of the sensor element. The sensor element here includes parts other than the sensing part (e.g., substrate, electrode, etc.). Non-Patent Document 1 and Non-Patent Document 2 describe that the reactivity of the probe molecule with the target molecule is not simply proportional to the amount of the probe molecule immobilized on the sensor element, and that if the amount of the probe molecule immobilized is too large, the reactivity with the target molecule may decrease due to steric hindrance. Since the reactivity of the probe molecule with the target molecule affects the sensitivity of the sensor, it can be said that the amount of the probe molecule immobilized is important for the sensing sensitivity.

[0005] Non-Patent Document 3 describes a method for analyzing the amount of immobilized probe molecules using ellipsometry, time-of-flight secondary ion mass spectrometry (TOF-SIMS) or atomic force microscope (AFM). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] MMLMVareiro,et al.,Anal.Chem.77,2426(2005). [Non-Patent Document 2] X. Zhao, et al., Langmuir 27, 7654 (2011). [Non-Patent Document 3] K. Gajos, et al., Applied Surface Science 518, 146269 (2020). Summary of the Invention [Problem to be solved by the invention]

[0007] However, the analysis method described in Non-Patent Document 3 has the following problems. Ellipsometry: Modeling is complex. TOF-SIMS: High vacuum is required, so there is a risk of the measurement sample being destroyed. Also, analysis is complicated. AFM: The measurement range is small, so it is only a few mm 2 This method is not suitable for wide-ranging evaluation because it takes a long time.

[0008] As described above, conventional methods for evaluating the amount of immobilized probe molecules have problems such as being unsuitable for wide-range evaluation, having complicated measurement or analysis methods, and having limitations on the composition, shape, etc. of the measurement sample.

[0009] The present invention has been made to solve the above problems, and aims to provide a sensor that can easily estimate the amount of immobilized probe molecules on a sensor element in a short time. It is also an object of the present invention to provide a detection method and a detection device using the above sensor. [Means for solving the problem]

[0010] The sensor of the present invention comprises a sensor element and a probe molecule immobilized on the surface of the sensor element, and the probe molecule is modified with a labeling substance for estimating the amount of the probe molecule immobilized on the sensor element.

[0011] The detection method of the present invention is a method for detecting a substance to be detected using the sensor of the present invention, and comprises the steps of: supplying a sample containing the substance to be detected to the sensor; measuring a physical quantity in the sensor that changes due to an interaction between the substance to be detected and a probe molecule of the sensor; and correcting the output value of the sensor obtained from the physical quantity based on the amount of immobilization of the probe molecule in the sensor estimated from the labeling substance modified to the probe molecule of the sensor, using a calibration curve that is prepared in advance from a known amount of the probe molecule and that shows the relationship between the amount of immobilization of the probe molecule and the reactivity of the probe molecule to the substance to be detected.

[0012] The detection device of the present invention is a device for detecting a substance to be detected using the sensor of the present invention, and comprises a measurement unit that measures a physical quantity in the sensor that changes due to interaction between the substance to be detected and a probe molecule of the sensor, and a correction unit that corrects the output value of the sensor obtained from the physical quantity based on the immobilization amount of the probe molecule in the sensor estimated from the labeling substance modified to the probe molecule of the sensor, using a calibration curve that is created in advance from a known amount of the probe molecule and shows the relationship between the immobilization amount of the probe molecule and the reactivity of the probe molecule to the substance to be detected. Effect of the Invention

[0013] According to the present invention, it is possible to provide a sensor capable of estimating the amount of immobilized probe molecules on a sensor element easily and in a short time. Furthermore, according to the present invention, it is possible to provide a detection method and a detection device using the above sensor. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram showing an example of a biosensor according to a first embodiment of the present invention. [Diagram 2] FIG. 2 is a schematic diagram showing an example of covalent bonding using an amino group of a probe molecule. [Diagram 3] FIG. 3 is a schematic diagram showing an example of covalent bonding using a sulfhydryl group of a probe molecule. [Figure 4] FIG. 4 is a schematic diagram showing an example of a biosensor according to the third embodiment of the present invention. [Diagram 5] FIG. 5 is a schematic diagram showing an example of a biosensor according to the fourth embodiment of the present invention. [Figure 6] FIG. 6 is a schematic diagram showing another example of the biosensor according to the fourth embodiment of the present invention. [Figure 7] FIG. 7 is a schematic diagram showing an example of a biosensor according to the fifth embodiment of the present invention. [Figure 8] FIG. 8 is a diagram showing an example of the structure of a linker molecule. [Figure 9] FIG. 9 is a schematic diagram showing an example of a biosensor according to the sixth embodiment of the present invention. [Figure 10] FIG. 10 is a schematic diagram showing an example of a biosensor according to the seventh embodiment of the present invention. [Figure 11] FIG. 11 is a schematic diagram showing an example of a state in which a biosensor is used. [Figure 12] FIG. 12 is a graph showing the relationship between the gate voltage VG and the source-drain current IDS. [Figure 13] FIG. 13 is an explanatory diagram of a process for modifying a probe molecule with a labeling substance. [Figure 14] FIG. 14 is an explanatory diagram of a process for producing a graphene FET device. [Figure 15] FIG. 15 is an explanatory diagram of a process for forming an oxide film. [Figure 16] FIG. 16 is an explanatory diagram of the process of carrying out the silane coupling treatment. [Figure 17]FIG. 17 is an explanatory diagram of the process of immobilizing labeled probe molecules. [Figure 18] FIG. 18 shows fluorescence micrographs obtained when the amount of labeled probe molecule added was varied. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The sensor, the detection method and the detection device of the present invention will be described below. However, the present invention is not limited to the following embodiments, and can be appropriately modified and applied within the scope of the present invention. The present invention also includes a combination of two or more of the individual preferable configurations of the present invention described in the following embodiments.

[0016] As an embodiment of the sensor of the present invention, a biosensor will be described as an example. The sensor of the present invention can also be applied to sensors other than biosensors. For example, the sensor of the present invention can be applied to sensors such as pH sensors and ion sensors.

[0017] The following embodiments are merely examples, and it goes without saying that partial replacement or combination of the configurations shown in the different embodiments is possible. From the second embodiment onwards, the description of the matters common to the first embodiment will be omitted, and only the differences will be described. In particular, the same effects and advantages due to the same configuration will not be mentioned one by one for each embodiment.

[0018] [First embodiment] FIG. 1 is a schematic diagram showing an example of a biosensor according to a first embodiment of the present invention.

[0019] In addition, the configuration of the biosensor has been appropriately modified in Fig. 1 for clarity and simplification of the drawing, and the same applies to the other drawings.

[0020] 1 includes a sensor element 10 and a probe molecule 20 immobilized on the surface of the sensor element 10. The probe molecule 20 is modified with a labeling substance 30. Thus, a labeled probe molecule 25 in which the probe molecule 20 is modified with the labeling substance 30 is immobilized on the surface of the sensor element 10.

[0021] In the sensor of the present invention, examples of the probe molecule immobilized on the surface of the sensor element include antibodies, enzymes, sugar chains, aptamers (nucleic acids), lectins, oligonucleotides, peptides, and low molecular weight organic polymers. Among these, at least one selected from the group consisting of antibodies, enzymes, peptides, and lectins is preferred. As long as the probe molecule remains on the surface of the sensor element, it may be movable with a certain degree of freedom.

[0022] In the sensor of the present invention, by detecting the labeling substance modified on the probe molecule, it is possible to estimate the amount of immobilized probe molecule on the sensor element in a short time and easily, as compared with the conventional evaluation method. Specifically, it is possible to expect the following effects: a wide range of evaluation can be performed in a short time, evaluation can be performed without setting complicated measurement conditions or performing analysis, and evaluation can be performed without processing the sample while maintaining a state that allows biosensing.

[0023] Furthermore, the probe molecule modified with the labeling substance has a larger molecular weight, which results in a stronger van der Waals force, and therefore the probe molecule is more likely to be immobilized on the surface of the sensor element.

[0024] In addition, depending on the type of labeling substance, it becomes possible to control the hydrophilicity or hydrophobicity of the surface of the probe molecule and the amount and sign of the charge, which makes it easier to capture the target molecule and increases the sensitivity.

[0025] For example, when a probe molecule is given hydrophilicity, non-specific adsorption is suppressed by hydrophilizing the surface, resulting in high sensitivity, whereas when a probe molecule is given hydrophobicity, hydrophobic interaction with the target molecule is enhanced, making it easier to capture the target molecule, resulting in high sensitivity.

[0026] In addition, by controlling the charge of the probe molecule, it is possible to increase the electrostatic attraction or decrease the electrostatic repulsion acting between the probe molecule and the target molecule, which makes it easier to capture the target molecule and increases the sensitivity.

[0027] [Second embodiment] In a second embodiment of the present invention, the labeling substance is modified onto the probe molecule by a covalent bond, which forms a stable bond, making it difficult for the labeling substance to be removed from the probe molecule.

[0028] For example, the amino group (-NH 2 ), sulfhydryl group (-SH), or carboxyl group (-COOH) can be used to covalently bond with a labeling substance. In this case, the functional group present at the end of the amino acid constituting the probe molecule may be used. For example, the amino group at the end of lysine, the sulfhydryl group at the end of cysteine, the carboxyl group at the end of aspartic acid or glutamic acid, etc. can be used.

[0029] FIG. 2 is a schematic diagram showing an example of covalent bonding using an amino group of a probe molecule.

[0030] As shown in FIG. 2, for example, a covalent bond is formed between an amino group of a probe molecule 20 and an active ester of a labeling substance 30 .

[0031] FIG. 3 is a schematic diagram showing an example of covalent bonding using a sulfhydryl group of a probe molecule.

[0032] As shown in FIG. 3, for example, a covalent bond is formed between a sulfhydryl group of a probe molecule 20 and a maleimide group of a labeling substance 30 .

[0033] When a carboxyl group of a probe molecule is used, the carboxyl group is activated with an activator such as EDC / NHS, thereby forming a covalent bond with an amino group of a labeling substance.

[0034] [Third embodiment] In a third embodiment of the present invention, the sensor element is a field effect transistor (FET) element, and the labeling substance is a fluorescent dye, a fluorescent protein, a quantum dot, or a gold nanoparticle.

[0035] FIG. 4 is a schematic diagram showing an example of a biosensor according to the third embodiment of the present invention.

[0036] The biosensor 2 shown in Fig. 4 includes a FET element 11 and a probe molecule 20 immobilized on the surface of the FET element 11. The probe molecule 20 is modified with a fluorescent dye or a fluorescent protein 31. Thus, a labeled probe molecule 25 in which the probe molecule 20 is modified with the fluorescent dye or the fluorescent protein 31 is immobilized on the surface of the FET element 11. Instead of the fluorescent dye or the fluorescent protein 31, the probe molecule 20 may be modified with quantum dots or gold nanoparticles 32 (see Fig. 5).

[0037] Fluorescent dyes, fluorescent proteins, and quantum dots can be observed using a fluorescence microscope, and gold nanoparticles can be observed using a dark-field microscope. Therefore, by modifying a probe molecule with a fluorescent dye, fluorescent protein, quantum dot, or gold nanoparticle as a labeling substance, it becomes possible to quickly and easily estimate the change over time in the amount of immobilized probe molecule using a fluorescence microscope, a dark-field microscope, or an optical microscope.

[0038] A FET-type biosensor forms a mechanism that mimics a living body in the channel portion, and detects the reaction that occurs there by the electrical properties of the FET. When the sensor element is an FET element, the doping amount and surface potential of the semiconductor can be adjusted by immobilizing a probe molecule modified with a labeling substance on the surface, making it possible to achieve high sensitivity.

[0039] For example, when the amino group of the probe molecule is modified, it is p-doped, and when the carboxyl group of the probe molecule is modified, it is n-doped.

[0040] In addition, the closer the surface potential is to 0 mV, the higher the sensitivity becomes. The surface potential can be measured with a zeta potential measuring device.

[0041] A fluorescent dye is a fluorescent label having a molecular weight of about 10,000 or less. Examples of fluorescent dyes include FITC (fluorescein isothiocyanate), Alexa Fluor (registered trademark) dye, and Cy (registered trademark) dye. A fluorescent protein is a fluorescent label having a molecular weight of about 10,000 or more. Examples of fluorescent proteins include PE (Phycoerythrin), APC (Allophycocyanin), and the like.

[0042] The size of the quantum dots is, for example, several nm or more and several tens of nm or less, and the particle diameter of the gold nanoparticles is, for example, several tens of nm or more and several hundreds of nm or less.

[0043] [Fourth embodiment] In a fourth embodiment of the invention, the sensor element is a quartz crystal microbalance (QCM) element or a surface plasmon resonance (SPR) element.

[0044] FIG. 5 is a schematic diagram showing an example of a biosensor according to the fourth embodiment of the present invention.

[0045] 5 includes a QCM element or SPR element 12 and a probe molecule 20 immobilized on the surface of the QCM element or SPR element 12. The probe molecule 20 is modified with quantum dots or gold nanoparticles 32. Thus, a labeled probe molecule 25 in which the probe molecule 20 is modified with the quantum dots or gold nanoparticles 32 is immobilized on the surface of the QCM element or SPR element 12.

[0046] When the sensor element is a QCM element or an SPR element, by modifying the probe molecule with quantum dots or gold nanoparticles as a labeling substance, it becomes possible to quickly and easily estimate the change over time in the amount of immobilized probe molecule using a fluorescence microscope, a dark-field microscope, or an optical microscope.

[0047] FIG. 6 is a schematic diagram showing another example of the biosensor according to the fourth embodiment of the present invention.

[0048] 6 includes a QCM element or an SPR element 12, and a probe molecule 20 immobilized on the surface of the QCM element or the SPR element 12. The probe molecule 20 is immobilized on the surface of the QCM element or the SPR element 12 via an insulating film 40. The probe molecule 20 is modified with a fluorescent dye or a fluorescent protein 31.

[0049] When the sensor element is a QCM element or an SPR element, by modifying the probe molecule with a fluorescent dye or fluorescent protein as a labeling substance, it becomes possible to quickly and easily estimate the change over time in the amount of immobilized probe molecule using a fluorescence microscope.

[0050] Furthermore, by providing an insulating film on the surface of the sensor element, it becomes possible to suppress quenching of fluorescence.

[0051] The insulating film may be, for example, an oxide film which will be described in a sixth embodiment below.

[0052] [Fifth embodiment] In a fifth embodiment of the present invention, the sensor element is a FET element, and the FET element includes graphene or a carbon nanotube. The probe molecule is immobilized on the surface of the FET element via a linker molecule including a pyrenyl group. The linker molecule includes a linker molecule having a benzene ring and capable of π-π interaction, in addition to the linker molecule including the pyrenyl group.

[0053] FIG. 7 is a schematic diagram showing an example of a biosensor according to the fifth embodiment of the present invention.

[0054] 7 includes a FET element 11 and a probe molecule 20 immobilized on the surface of the FET element 11. The probe molecule 20 is modified with a labeling substance 30. Thus, a labeled probe molecule 25 in which the probe molecule 20 is modified with the labeling substance 30 is immobilized on the surface of the FET element 11.

[0055] The FET element 11 includes an insulating substrate 50, a semiconductor layer 51 disposed on the insulating substrate 50, and a source electrode 52 and a drain electrode 53 disposed on the insulating substrate 50 and electrically connected to the semiconductor layer 51. The source electrode 52 and the drain electrode 53 are disposed on the insulating substrate 50 away from each other, and the insulating substrate 50 is exposed between the source electrode 52 and the drain electrode 53. In the example shown in FIG. 7, the semiconductor layer 51 is disposed on the insulating substrate 50 so as to cover the exposed portion of the insulating substrate 50. The semiconductor layer 51 may be disposed on the insulating substrate 50 so as to cover an end of the source electrode 52, an exposed portion of the insulating substrate 50, and an end of the drain electrode 53. The semiconductor layer 51 between the source electrode 52 and the drain electrode 53 constitutes a channel of the FET element 11.

[0056] The insulating substrate 50 is, for example, a silicon (Si) substrate having a surface oxidized to form a silicon oxide (SiO 2Examples of the insulating substrate 50 include a thermally oxidized silicon substrate on which a thermally oxidized silicon (Si) layer is formed, a boron nitride (BN) substrate, etc. The material of the insulating substrate 50 is not particularly limited, and examples thereof include inorganic compounds such as silicon oxide, silicon nitride, aluminum oxide, titanium oxide, and calcium fluoride, and organic compounds such as acrylic resin, polyimide, and fluororesin. The shape of the insulating substrate 50 is not particularly limited, and may be a flat plate or a curved plate. The insulating substrate 50 may be flexible.

[0057] The semiconductor layer 51 includes graphene or carbon nanotubes. By using a FET transistor with a channel made of graphene or carbon nanotubes, it is possible to increase the sensitivity of the sensor.

[0058] Graphene is a two-dimensional material made of carbon atoms bonded in a hexagonal network that has a very large specific surface area (surface area per volume) and very high electrical mobility.

[0059] Generally, graphene refers to a carbon-based sheet-like material in which one layer of carbon atoms has a honeycomb structure. However, in this specification, the following materials are also broadly defined as graphene. A carbon-based sheet material in which graphene is multilayered or partially multilayered up to 100 layers. A carbon-based sheet material that is polycrystalline, has grain boundaries, is partially torn, and has edges. Carbon-based sheet materials in which elements have been partially substituted or the honeycomb structure has been destroyed. Graphene oxide and reduced graphene oxide obtained by reducing graphene oxide. - Ribbon-shaped (strip-shaped) graphene. Carbon nanotubes are made of sheets of graphene shaped into a cylinder, and are materials in which graphene is wrapped around itself.

[0060] Carbon nanotubes are long, cylindrical carbon compounds. For example, single-wall carbon nanotubes (SW-CNTs) are used, which have a single carbon layer with a mesh structure similar to that of graphene.

[0061] The number of layers in the semiconductor layer 51 is not limited to one layer, and may be two or three or more layers. The number of layers in the semiconductor layer 51 is preferably ten or less, and more preferably five or less. The number of layers does not need to be uniform throughout the semiconductor layer 51, and may include, for example, a mixture of one layer and two or more layers. The number of layers in the semiconductor layer 51 can be measured, for example, by Raman spectroscopy or cross-sectional observation using a transmission electron microscope (TEM).

[0062] The source electrode 52 and the drain electrode 53 are electrodes with a multilayer structure in which, for example, a titanium (Ti) layer and a gold (Au) layer are laminated. As the electrode material, in addition to titanium and gold, metals such as gold, platinum, titanium, and palladium may be used in a single layer, or two or more metals may be combined to be used as a multilayer structure.

[0063] A linker molecule 60 is disposed on the surface of the semiconductor layer 51. The probe molecule 20 is immobilized on the surface of the FET element 11 via the linker molecule 60 that includes a pyrenyl group.

[0064] FIG. 8 is a diagram showing an example of the structure of a linker molecule.

[0065] FIG. 8 shows 1-pyrenebutanoic acid succinimidyl ester (PBASE) as an example of a linker molecule containing a pyrenyl group.

[0066] As shown in Figure 8, one end of PBASE has a succinimide group, so that, for example, a linker molecule can be bonded to the amino group of a probe molecule to immobilize the probe molecule. In addition, the other end of PBASE has a pyrenyl group with four benzene rings arranged in a plane, so that it can be bonded to the surface of a carbon-based semiconductor layer such as graphene by a non-covalent bond called π-π interaction. Therefore, compared to when the probe molecule is placed on the surface of the semiconductor layer by a covalent bond, the effect on the crystal structure of the semiconductor layer can be reduced, and deterioration of characteristics can be suppressed.

[0067] Furthermore, by detecting the labeling substance modified on the probe molecule, the presence or absence of breakage in the carbon-based semiconductor layer can be confirmed. In particular, when the labeling substance is a fluorescent dye, a fluorescent protein, a quantum dot, or a gold nanoparticle, the presence or absence of breakage in the carbon-based semiconductor layer can be visually confirmed.

[0068] [Sixth embodiment] In a sixth embodiment of the present invention, the sensor element is an FET element, and the FET element includes graphene or carbon nanotubes. The probe molecule is immobilized on the surface of the FET element via an oxide film. The oxide film covers the surface of the semiconductor layer, making the surface of the semiconductor layer hydrophilic. This suppresses non-specific adsorption, resulting in high sensitivity.

[0069] In the sixth embodiment of the present invention, the probe molecule is preferably immobilized on the surface of the FET element via a silane coupling agent present on the surface of the oxide film. When the silane coupling agent is present on the surface of the oxide film, the oxide film and the silane coupling agent, and the silane coupling agent and the probe molecule are firmly bonded by covalent bonds, respectively. Therefore, the probe molecule is stably immobilized, resulting in high sensitivity.

[0070] FIG. 9 is a schematic diagram showing an example of a biosensor according to the sixth embodiment of the present invention.

[0071] In the biosensor 6 shown in Fig. 9, an oxide film 61 is disposed on the surface of a semiconductor layer 51, and a silane coupling agent 62 is disposed on the surface of the oxide film 61. The probe molecule 20 modified with the labeling substance 30 (i.e., the labeled probe molecule 25) is immobilized on the surface of the FET element 11 via the oxide film 61 and the silane coupling agent 62. Note that the silane coupling agent 62 does not have to be disposed on the surface of the oxide film 61, and in that case, it is sufficient that the probe molecule 20 modified with the labeling substance 30 (i.e., the labeled probe molecule 25) is immobilized on the surface of the FET element 11 via the oxide film 61.

[0072] 9, oxide film 61 is disposed over the entire surface of insulating substrate 50 so as to cover the surface of semiconductor layer 51. As shown in FIG. 9, oxide film 61 may also be disposed over source electrode 52 and drain electrode 53, but it is sufficient that oxide film 61 is disposed over insulating substrate 50 so as to cover at least the surface of semiconductor layer 51.

[0073] The oxide constituting the oxide film 61 is, for example, SiO 2 , Al 2 O 3 , TiO 2 , HfO 2 , ZrO 2 , SiN x Alternatively, a composite oxide thereof may be used.

[0074] The oxides constituting the oxide film 61 can be confirmed by performing elemental analysis on the sensor surface using X-ray photoelectron spectroscopy (XPS) or by performing elemental analysis on the sensor surface using energy dispersive X-ray spectroscopy (EDS).

[0075] Examples of methods for forming the oxide film 61 include evaporation, sputtering, atomic layer deposition (ALD), thermal chemical vapor deposition (thermal CVD), and catalytic chemical vapor deposition (catalytic CVD).

[0076] From the viewpoint of ensuring electrical insulation of the sensor surface and ensuring mechanical stability of the oxide film 61 (for example, mechanical stability against ultrasonic cleaning), the thickness of the oxide film 61 is preferably 2 nm or more. On the other hand, the thickness of the oxide film 61 is preferably 30 nm or less. If the thickness of the oxide film 61 is 30 nm or less, high sensitivity of the sensor is ensured.

[0077] The thickness of the oxide film 61 can be measured by observing a cross section with a transmission electron microscope (TEM).

[0078] The oxide film 61 preferably contains an amorphous material. This can improve the electrical insulation of the sensor surface compared to when the entire oxide film 61 is crystalline. When the oxide film 61 contains an amorphous material, the entire oxide film 61 does not necessarily have to be amorphous, and may partially contain crystalline regions.

[0079] The fact that the oxide film 61 contains an amorphous substance can be confirmed by performing a crystallinity analysis from an X-ray diffraction image or an electron beam diffraction image in a transmission electron microscope (TEM) measurement.

[0080] When a silane coupling agent 62 is present on the surface of the oxide film 61, examples of the silane coupling agent 62 include silane coupling agents having an amino group such as 3-aminopropyltriethoxysilane (APTES) and 3-aminopropyltrimethoxysilane (APTMS), silane coupling agents having a thiol group such as 3-mercaptopropyltriethoxysilane (MPTES), and silane coupling agents having an epoxy group such as triethoxy(3-glycidyloxypropyl)silane (GPTES).

[0081] The presence of the silane coupling agent 62 on the surface of the oxide film 61 can be confirmed by performing a surface analysis using time-of-flight secondary ion mass spectrometry (TOF-SIMS).

[0082] The silane coupling agent 62 can be replaced with another material as long as the material forms a covalent bond on the oxide film 61. Specific examples of such materials include phosphonic acid derivatives.

[0083] In the biosensor according to the sixth embodiment of the present invention, the spacer molecules may be immobilized on the surface of the FET element via the spacer molecules present on the surface of the oxide film.

[0084] When spacer molecules are present on the surface of the oxide film, the spacer molecules allow the probe molecules to separate from the surface of the oxide film and obtain a degree of freedom, improving the sensing ability of the probe molecules. In addition, when the spacer molecules are hydrophilic, the hydrophilicity of the sensor surface can be enhanced.

[0085] Examples of the spacer molecule include polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), dextran, ethylene glycol bis(succinimidyl succinate), etc. The arm length of the spacer molecule is preferably 0.7 nm or more and 10 nm or less, but is not limited thereto.

[0086] In the biosensor according to the sixth embodiment of the present invention, a seed layer may be provided between the semiconductor layer and the oxide film. By providing the seed layer, the oxide film is formed uniformly, thereby improving the sensitivity.

[0087] The seed layer can be formed by depositing a film of a single metal, such as a light metal such as aluminum (Al) or magnesium (Mg), a 3d transition metal such as titanium (Ti), nickel (Ni), or chromium (Cr), or a rare metal such as hafnium (Hf), zirconium (Zr), or yttrium (Y), and then oxidizing it.

[0088] The seed layer preferably has a thickness of 2 nm or less, whereas the seed layer preferably has a thickness of 0.5 nm or more.

[0089] In the biosensor according to the sixth embodiment of the present invention, the oxide film may have an uneven surface. The uneven surface of the oxide film can increase the surface area of ​​the oxide film, thereby enhancing the hydrophilicity of the sensor surface. In addition, the density of the probe molecules can be increased, resulting in high sensitivity.

[0090] Methods for forming irregularities on the surface of an oxide film include, for example, a method of roughening the surface by surface blasting or plasma ashing, and a method of island growing of an oxide film. Island growth is a phenomenon in which nuclei that are separated from each other start to grow. Since the film is formed non-uniformly due to island growth, irregularities are formed on the surface of the film. Requirements for island growth include poor wettability of the base surface of the film to be grown with respect to the raw material of the film.

[0091] [Seventh embodiment] In the seventh embodiment of the present invention, a blocking agent is present in the FET element together with the probe molecules. The blocking agent enhances the hydrophilicity of the sensor surface. As a result, non-specific adsorption is suppressed, resulting in high sensitivity.

[0092] FIG. 10 is a schematic diagram showing an example of a biosensor according to the seventh embodiment of the present invention.

[0093] 10, a labeled probe molecule 25, in which a probe molecule 20 is modified with a labeling substance 30, is immobilized on the surface of an FET element 11. In the FET element 11, a blocking agent 63 is present together with the probe molecule 20.

[0094] Examples of the blocking agent 63 include proteins (e.g., bovine serum albumin (BSA), hemoglobin, skim milk, etc.), surfactants (e.g., Tween (trade name), Triton (trade name), sodium dodecyl sulfate (SDS), etc.), and polymers (e.g., PEG, PVP, etc.). These blocking agents may be used alone or in combination of two or more.

[0095] In the sensor of the present invention, when the sensor element is a FET element, the FET element may further include a gate electrode for applying an electric field to the semiconductor layer from the outside.

[0096] The FET element operates, for example, in a liquid, and in this case, the surface of the FET element that comes into contact with the liquid has sites that can bind to a substance to be detected.

[0097] FIG. 11 is a schematic diagram showing an example of a state in which a biosensor is used.

[0098] The biosensor 100 shown in FIG. 11 includes an FET element 11 and a probe molecule 20 immobilized on the surface of the FET element 11, similar to the biosensor 6 shown in FIG. 9. The probe molecule 20 is modified with a labeling substance 30. The FET element 11 includes an insulating substrate 50, a semiconductor layer 51 disposed on the insulating substrate 50, and a source electrode 52 and a drain electrode 53 disposed on the insulating substrate 50 and electrically connected to the semiconductor layer 51. An oxide film 61 is disposed on the surface of the semiconductor layer 51, and a silane coupling agent 62 is disposed on the surface of the oxide film 61. The probe molecule 20 modified with the labeling substance 30 (i.e., the labeled probe molecule 25) is immobilized on the surface of the FET element 11 via the oxide film 61 and the silane coupling agent 62.

[0099] 11, a biosensor 100 is configured by attaching a pool 71 made of, for example, silicone rubber onto the FET element 11, filling the inside of the pool 71 with an electrolyte solution 72, immersing the gate electrode 54 in the electrolyte solution 72, and connecting a bipotentiostat (not shown) to the source electrode 52, the drain electrode 53, and the gate electrode 54. The electrolyte solution 72 contains a substance (target molecule) 73 to be detected.

[0100] The gate electrode 54 applies a potential to the source electrode 52 and the drain electrode 53, and generally uses a noble metal. The gate electrode 54 is provided at a position other than the positions where the source electrode 52 and the drain electrode 53 are formed. Usually, the gate electrode 54 is provided on the insulating substrate 50 or at a position other than the insulating substrate 50, but it is preferable to provide the gate electrode 54 above the source electrode 52 or the drain electrode 53.

[0101] Figure 12 shows the gate voltage V G and the source-drain current I DS 4 is a graph showing the relationship between

[0102] In Fig. 12, the source-drain current I DS is shown by solid line A, and the source-drain current I DS is indicated by dashed line B. As shown in Figure 12, when the probe molecule specifically binds to the substance to be detected, the conduction characteristics are modulated by the charge of the target molecule, which is the substance to be detected. By observing this modulation, it is possible to sense the presence or concentration of the substance to be detected.

[0103] The sensor of the present invention is not limited to a sensor element that electrically detects specific binding of a probe molecule to a substance to be detected, and a sensor element that optically or mechanically detects this may also be used.

[0104] The sensor of the present invention is not limited to the above-described embodiment, and various applications and modifications can be made within the scope of the present invention with respect to the configuration of the sensor, manufacturing conditions, and the like.

[0105] For example, in the sensor of the present invention, an insulating coating layer may be provided on a portion of the sensor element other than the sensing portion. By providing the insulating coating layer, the insulation of the portion other than the sensing portion is improved, thereby improving the reliability of the sensor. In addition, the sensitivity of the sensor is improved because the target molecules are not captured by the portion other than the sensing portion.

[0106] Examples of materials constituting the insulating coating layer include organic compounds such as polyimide, epoxy resin, acrylic resin, fluororesin, etc. The thickness of the insulating coating layer is preferably 100 nm or more and 10 μm or less.

[0107] Alternatively, when the sensor element is an FET element, an insulating coating layer may be provided on the source electrode and the drain electrode, and a semiconductor layer may be disposed on the source electrode, the drain electrode, and the insulating coating layer.

[0108] A method for detecting a target substance using the sensor of the present invention will be described below. Such a detection method also constitutes the present invention.

[0109] Before the measurement, the amount of immobilized probe molecules on the sensor is estimated by detecting the labeling substance modified on the probe molecules of the sensor. Also, a calibration curve showing the relationship between the amount of immobilized probe molecules and the reactivity of the probe molecules with the substance to be detected is prepared in advance from known amounts of the probe molecules.

[0110] A sample containing a substance to be detected is supplied to the sensor. This brings the sample into contact with the sensor element. For example, when the sample containing the substance to be detected is a liquid, the sample may be dropped onto the sensor element using a dropper or the like, or the sample may be introduced into the sensor element using a flow path.

[0111] Examples of samples containing a substance to be detected include body fluids such as saliva, throat swabs, nasal mucus, tears, and blood of a subject, biological samples such as urine and feces, suspensions of cells or viruses themselves, drinking water, sewage, breath, etc. A sample containing a substance to be detected does not have to be a liquid.

[0112] Next, in the sensor, a physical quantity that changes due to an interaction between the substance to be detected and the probe molecules of the sensor is measured.

[0113] The physical quantity to be measured varies depending on the type of sensor element. Examples of the physical quantity include electrical physical quantities and optical physical quantities. Examples of the electrical physical quantity include voltage value, current value, frequency, electrical resistance value, and conductivity. Examples of the optical physical quantity include light wavelength, light intensity, light reflectance, and light transmittance.

[0114] For example, when the sensor element is an FET element, a voltage is applied between the source electrode and the drain electrode to measure the value of a current (drain current value) flowing between the source electrode and the drain electrode.

[0115] Then, using a calibration curve, which has been prepared in advance from known amounts of probe molecules and shows the relationship between the amount of immobilized probe molecules and the reactivity of the probe molecules with the substance to be detected, the sensor output value obtained from the physical quantity is corrected based on the amount of immobilized probe molecules in the sensor estimated from the labeling substance modified on the probe molecules of the sensor.

[0116] As described above, there is a correlation between the amount of immobilized probe molecules on the sensor element and the reactivity of the probe molecules to the target substance. Therefore, a calibration curve showing the relationship between the amount of immobilized probe molecules and the reactivity of the probe molecules to the target substance is prepared in advance from a known amount of probe molecules. Using this calibration curve, the output value of the sensor obtained from the physical quantity, i.e., the signal response amount, can be corrected based on the amount of immobilized probe molecules in the sensor estimated from the labeling substance modified on the probe molecules of the sensor. Therefore, the accuracy and reliability of the measurement can be improved.

[0117] Furthermore, an apparatus for detecting a target substance using the sensor of the present invention also constitutes the present invention.

[0118] That is, the detection device of the present invention is a device for detecting a substance to be detected using the sensor of the present invention, and comprises a measurement unit that measures a physical quantity in the sensor that changes due to interaction between the substance to be detected and a probe molecule of the sensor, and a correction unit that corrects the output value of the sensor obtained from the physical quantity based on the immobilization amount of the probe molecule in the sensor estimated from the labeling substance modified to the probe molecule of the sensor, using a calibration curve that is created in advance from a known amount of the probe molecule and shows the relationship between the immobilization amount of the probe molecule and the reactivity of the probe molecule to the substance to be detected.

[0119] As in the detection method of the present invention, the amount of immobilized probe molecules on the sensor is estimated by detecting the labeling substance modified on the probe molecules of the sensor before measurement. The estimation of the amount of immobilized probe molecules is preferably performed using an apparatus other than the detection apparatus of the present invention. In addition, a calibration curve showing the relationship between the amount of immobilized probe molecules and the reactivity of the probe molecules with the substance to be detected is prepared in advance from known amounts of probe molecules. EXAMPLES

[0120] Hereinafter, examples will be given that more specifically disclose a biosensor, which is one embodiment of the sensor of the present invention, although the present invention is not limited to these examples.

[0121] First, a biosensor having a configuration similar to that of the biosensor 6 shown in FIG. 9 was fabricated by the following procedure.

[0122] (1) Labeling of probe molecules 13 is an explanatory diagram of a process for modifying a probe molecule with a labeling substance. As shown in FIG 13, a labeled probe molecule 25 is prepared by modifying a probe molecule 20 with a labeling substance 30 through a chemical bond.

[0123] The specific conditions are as follows:

[0124] Probe molecule: Anti-human CRP monoclonal antibody (Oriental Yeast Co., Ltd.) or Anti-Influenza A Virus Nucleoprotein monoclonal antibody (BioMatrix Research Institute, Inc.) Labeling substance: Alexa Fluor 488 NHS Ester (Thermo Fisher Scientific)

[0125] (Labeling Procedure) 1. Dissolve the above labeling substance (fluorescent dye) in N,N-dimethylformamide (DMF) to prepare a 10 mg / mL solution. 2. Add the solution prepared in step 1 dropwise to 1 to 10 mg / mL of the above probe molecule (antibody), and react with stirring at room temperature for 1 hour. 3. Remove unreacted labeled substance using a desalting column. 4. Add a preservative (sodium azide) to the solution after modification with the labeling substance and store at 4°C.

[0126] (2) Fabrication of graphene FET devices 14 is an explanatory diagram of a process for producing a graphene FET element. As shown in FIG 14, a semiconductor layer 51, a source electrode 52, and a drain electrode 53 are formed on an insulating substrate 50, to produce a graphene FET element 15.

[0127] For example, a Ti layer and an Au layer are formed on the insulating substrate 50 using a method such as vacuum deposition, electron beam (EB) deposition, or sputtering. Then, the source electrode 52 and the drain electrode 53 are formed by patterning using photolithography and etching. Next, graphene grown on, for example, a copper foil is transferred to the insulating substrate 50, and then, the semiconductor layer 51 is formed on the insulating substrate 50 by patterning using photolithography and etching.

[0128] (3) Formation of oxide film 15 is an explanatory diagram of a process for forming an oxide film. As shown in FIG.

[0129] For example, an oxide film 61 made of silica, alumina or a composite oxide thereof is formed by using a method such as atomic layer deposition (ALD) or EB evaporation.

[0130] (4) Silane coupling treatment 16 is an explanatory diagram of a process of performing a silane coupling treatment. As shown in FIG 16, the surface of an oxide film 61 is subjected to a silane coupling treatment.

[0131] The silane coupling treatment can be carried out by a gas phase method or a liquid phase method. In the example shown in Fig. 16, GPTES is used as the silane coupling agent 62, and an epoxy group is present on the surface.

[0132] (5) Immobilization of labeled probe molecules 17 is an explanatory diagram of a process for immobilizing a labeled probe molecule. As shown in FIG. 17, a labeled probe molecule 25 is immobilized on the surface of a graphene FET element 15.

[0133] For example, the solution of the labeled probe molecules 25 prepared in (1) is dropped onto the graphene FET element 15 that has been subjected to the silane coupling treatment in (4).

[0134] The biosensor thus prepared was observed under a fluorescent microscope under the following observation conditions: field of view: 500 μm, objective lens: 20x, exposure time: 1 to 5 seconds.

[0135] FIG. 18 shows fluorescence micrographs obtained when the amount of labeled probe molecule added was varied.

[0136] As can be seen from Figure 18, when the amount of labeled probe molecule added is 0 nM, no fluorescence is observed, whereas when the amount of labeled probe molecule added is 67 nM or 667 nM, fluorescence is observed, and it can be confirmed that the fluorescence becomes stronger as the amount added increases.

[0137] These results confirmed that in biosensors in which a fluorescent dye is used as a labeling substance to modify the probe molecule, the amount of immobilized probe molecule can be estimated by detecting the labeling substance using a fluorescence microscope. [Explanation of symbols]

[0138] 1, 2, 3, 4, 5, 6, 7, 100 Biosensors 10 Sensor element 11 FET element 12 QCM element or SPR element 15 Graphene FET device 20 Probe molecules 25 Labeled probe molecules 30 Labeled substance 31 Fluorescent dyes or fluorescent proteins 32 Quantum dots or gold nanoparticles 40 Insulating film 50 Insulating substrate 51 Semiconductor layer 52 Source electrode 53 Drain electrode 54 Gate electrode 60 Linker Molecules 61 Oxide Film 62 Silane coupling agents 63 Blocking Agents 71 Pool 72 Electrolyte 73 Detected substance (target molecule)

Claims

1. A sensor element; A probe molecule immobilized on a surface of the sensor element, the probe molecule is modified with a labeling substance for estimating the amount of the probe molecule immobilized on the sensor element; The sensor, wherein the sensor element is a field effect transistor (FET) element.

2. The sensor according to claim 1 , wherein the labeling substance is modified onto the probe molecule by covalent bonding.

3. The sensor according to claim 1 or 2, wherein the labeling substance is a fluorescent dye, a fluorescent protein, a quantum dot, or a gold nanoparticle.

4. The sensor of claim 1 , wherein the FET element comprises graphene or a carbon nanotube.

5. The sensor according to claim 4 , wherein the probe molecule is immobilized on the surface of the FET element via a linker molecule containing a pyrenyl group.

6. 5. The sensor according to claim 4, wherein the probe molecule is immobilized on the surface of the FET element via an oxide film.

7. 7. The sensor according to claim 6, wherein the probe molecule is immobilized on the surface of the FET element via a silane coupling agent present on the surface of the oxide film.

8. The sensor according to any one of claims 1 to 7, wherein a blocking agent is present in the FET element together with the probe molecule.

9. 9. The sensor according to claim 1, wherein the probe molecule is at least one selected from the group consisting of an antibody, an enzyme, a peptide, and a lectin.

10. A method for detecting a target substance using the sensor according to any one of claims 1 to 9, comprising: supplying a sample containing the target substance to the sensor; a step of measuring, in the sensor, an electrical physical quantity that changes due to an interaction between the substance to be detected and a probe molecule of the sensor; and correcting an output value of the sensor obtained from the electrical physical quantity based on the amount of immobilization of the probe molecule in the sensor estimated from the labeling substance modified on the probe molecule of the sensor, using a calibration curve, which is created in advance from a known amount of the probe molecule and shows the relationship between the amount of immobilization of the probe molecule and the reactivity of the probe molecule with the substance to be detected.

11. An apparatus for detecting a target substance using the sensor according to any one of claims 1 to 9, comprising: a measurement unit in the sensor that measures an electrical physical quantity that changes due to an interaction between the substance to be detected and a probe molecule of the sensor; a correction unit that corrects an output value of the sensor obtained from the electrical physical quantity based on the amount of immobilization of the probe molecule in the sensor estimated from the labeling substance modified to the probe molecule of the sensor, using a calibration curve that is created in advance from a known amount of the probe molecule and that shows the relationship between the amount of immobilization of the probe molecule and the reactivity of the probe molecule with the substance to be detected.

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